Tissue repair or "wound healing" involves many cellular and biochemical reactions which transform quiescent connective tissue and epidermal cells into rapidly dividing, rapidly moving cells. This transformation involves chemotaxis (movement of cells), mitogenesis (division of cells), and increased protein synthesis. Epidermal cells, fibroblasts, and capillary endothelial cells are involved in the formation of new tissue. Epidermal cells migrate and divide to form new skin (epithelium) at the site of tissue repair; fibroblasts move and divide to produce the matrix which fills the repair site (granulation site); capillary endothelial cells migrate and divide to produce new capillaries which revascularize the fibroblast/collagen matrix.
The process of cellular migration and mitosis is under the control of several biochemical agents. These locally-acting agents act on the various cells to direct their movement and division.
Each of these biochemical agents is (1) chemotactic (i.e., a chemoattractant) and causes migration (chemotaxis) of a certain type of cell; (2) mitogenic (i.e., a mitogen) and causes division (mitogenesis) of a certain type of cell; and/or (3) angiogenic (i.e., an angiogenic factor) and causes formation of new capillaries (migration and division of capillary endothelial cells). The chemotactic, mitogenic or angiogenic character of a biochemical agent is generally determined by testing the agent in various known assays for chemotaxis, mitogenesis and anglogenesis. Some of these assays are described below. Additional assays are expected to be developed in the future which will be directed at the same characteristics but perhaps will more clearly define or measure the presence of the characteristic.
Platelet-Derived Growth Factor (PDGF) is a well-characterized, 30,000 dalton dimeric glycoprotein with mitogenic and chemoattractant activity for fibroblasts, smooth muscle cells and glial cells. In the presence of PDGF, fibroblasts move into the area of tissue needing repair and are stimulated to divide in the wound space itself. The cells exposed to lower PDGF concentrations (approximately 0.5-1 ng/ml) are stimulated to move and, as they move to environments having higher concentrations of PDGF (approximately 1-2 ng/ml), they are stimulated to divide.
The process of neovascularization or angiogenesis (new capillary formation) is stimulated by angiogenesis factors. The angiogenesis factor recoverable from platelets (PDAF) is a pure chemoattractant without mitogenic activity for capillary endothelial cells, which stimulates them to move through a gradient towards the source of the angiogenesis factor. Once the capillary cells start to move from the parent capillaries, they start to divide. This division is probably under the control of autocrine growth factors produced by endothelial cells, which have been found to be of the basic fibroblast growth factor (FGF) variety.
The combination of fibroblast migration and mitosis and endothelial cell migration and mitosis produces granulation tissue. Granulation tissue also is rich in neutrophils and monocytes which have been brought to the site of repair by the presence of C5A from complement activation and transforming growth factor beta (TGF-B) from platelets. The presence of these phagocytic cells decreases the contamination and prevents overt infection.
TGF-B is a 25,000 dalton (112 amino acid) polypeptide that has a function in the synthesis of fibrin-collagen. TGF-B inhibits the division of fibroblasts and increases their matrix production. Whether TGF-B stimulates or inhibits division is a function of the entire set of growth factors operating in the tissue. In the presence of PDGF, TGF-B usually stimulates division while it usually inhibits division in the presence of epidermal growth factors (described below).
After granulation tissue is formed, epidermal cells migrate from the cut skin edge over the granulation tissue to form a new skin layer, which then matures into normal skin. This cellular activity is at least partially under the control of platelet-derived epidermal growth factor (PDEGF) which is a chemoattractant for epidermal cells.
In summary, the process of tissue repair or "wound healing" is under the control of at least four growth factors: TGF-B, PDGF, PDAF and PDEGF. The presence of these growth factors in the tissue to be repaired produce fibroblast migration and mitosis, endothelial cell migration and subsequent mitosis, and epidermal migration and mitosis. The end result is the filling of wound space with a granulation tissue followed by reepithelialization and skin maturation.
Two principal sources of these factors for natural healing of tissue are platelets and macrophages. When tissue is damaged in the body, platelets are released by the presence of thrombin generated by the activation of the coagulation process. These platelets then release PDGF, PDAF, PDEGF, TGF-B and platelet factor 4 (PF-4 is a chemoattractant for neutrophils and monocytes) and stimulate release of complement C5A. The PDGF, PDAF, PDEGF and PF-4 themselves contribute directly to healing the wound as described above, while TGF-B and C5A attract macrophages to the damaged site. Macrophages also release same or similar mitogens, chemoattractants, and angiogenesis factors once they have been summoned to the area of tissue repair. However, to date, macrophages are not known to produce EGF-like activity.
Common reasons for the failure of nonhealing wounds to improve are: infection, poor cellularity, few fibroblasts, no new capillaries and few inflammatory cells. In contrast, healing wounds are characterized by mononuclear and macrophage cell infiltrates, dividing fibroblasts and numerous capillaries.
Knighton, et al, Ann. Sur. 1986, 204:322-330, incorporated in its entirety herein by reference thereto, treated 49 patients with chronic nonhealing cutaneous ulcers using autologous Platelet Derived Wound Healing Formula (PDWHF) in a microcystalline collagen salve. The wounds had been treated an average of 198 weeks with conventional treatment. Mean healing time to 100% epithelization was 10.6 weeks direct correlation to 100% healing was related to initial wound size and initiation of PDWFIF therapy. There was no abnormal tissue formation, keloid or hypertrophic scarring reported.
In a double-blind study, Knighton et al, Tissue Repair Symposium at Tarpon Springs, Florida May 1987, which is incorporated herein in its entirety by reference thereto, compared wound healing using PDWHF in a collagen base to a placebo. All 24 patients received wound care according to a standard protocol. The 13 patients in the PDWHF group had healing to 100% epithelization in 17 or 21 wounds after 8 weeks of therapy; of 11 patients in the placebo group only 2 of 13 had wounds that reached 100% epithelization. The placebo failures were then treated with PDWHF and their wounds healed in an average of 7.1 weeks. The unhealed PDWHF treated patients continued on PDWHF and achieved 100% epithelization in an average of 5.8 additional weeks of treatment.
The pretreatment records for a group of patients seen at the University of Minnesota Wound Care Clinic were provided to a panel of three nationally recognized experts in amputation, diabetic foot care and vascular surgery. The 136 wounds in 73 patients were graded on a severity scale of 1 through 6 (partial thickness to full thickness with gangrene). PDWHF therapy had been initiated in each case. Over 75% had wound grades of 3 or higher. Seventy (70%) of the limbs evaluated were considered to be at significant or definite risk of amputation. Of the 26 limbs scored at no risk of amputation, none required amputation. Over 90% of the 53 limbs considered to be at significant risk of amputation were salvaged. Limbs scored at requiring immediate amputation (n=9) had a salvage rate of 86%. Applying PDWHF to wounds on a daily basis promoted granulation tissue formation and epidermal cell growth stimulation. Chronic nonhealing wounds have been healed to fully functional skin.
In the foregoing studies of wound treatment, the PDWHF formulation was based on releasing 10.sup.9 platelets into medium to a final volume of 1 ml. No attempt was made to adjust the amount of platelet releasate to compensate for variation from donor to donor, or from time to time for a particular donor, of the potencies of the wound healing factors contained in such platelet releasate. It is an object of the present invention to select amounts of platelet releasate for efficacious treatment of tissue, giving due regard to variations of the potencies of healing factors contained in the releasate.